The object of this study is to carry out a functional analysis of the Herpes simplex virus type-common envelope glycoprotein D. We cloned the gene for gD-1 (oral) into a M13-E. coli expression system. Large amounts of the gD-1 and gD-2 protein are synthesized, and three sequential epitopes are found in the immunosorbant purified expressed protein. These epitopes were localized precisely and monoclonal antibodies to two of these epitopes (Group 11 and VII) protect mice by passive immunization from a lethal virus challenge. Oligonucleotide site-directed mutagenesis studies are being directed at altering a base in the position of the gene that codes for the predicted beta turn in one epitope. None of the four conformational epitopes are present in the M13-E. coli expressed gD. Concurrently, we cloned the gene, including the region for the signal into Prsv-o, a eucaryotic expression vector. Full sized gD-1 is transiently expressed, as well as a 20K fragment. This fragment which represents the N-terminus of gD is secreted into the medium and represents the shortest piece of gD so far synthesized. This fragment, as well as others, will be used to localize further the conformational epitopes. Experiments are in progress to develop permanent 3T3 cell lines using a hygromycin resistant plasmid. Immunosorbent purified gD protects mice against an IP or foot-pad HSV-2 challenge when administered either IP or IM and in the presence of complete Freunds adjuvent or alum. Synthetic peptides mimicking different configurations of the first 23 amino acids of gD also induce solid protection in mice against death and paralysis.